Melittin对非小细胞肺癌增殖、凋亡及PI3K/Akt信号通路的影响

目的探讨Melittin对非小细胞肺癌（NSCLC）细胞增殖、凋亡及PI3K/Akt信号通路的影响。方法分别采用0、10、20、50、100μmol/L Melittin处理NSCLC细胞株A549、SPC-A1及人肺上皮细胞株16HBE 24、48、72和96 h后,采用四甲基偶氮唑盐（MTT）比色法检测各细胞株的增殖抑制率变化,同时采用流式细胞术Annexin-FITC/PI双染法及PI单染法检测不同浓度Melittin处理24、48 h后的A549细胞凋亡及细胞周期情况,Western blotting检测不同浓度Melittin处理48 h后的A549细胞中PI3K/Akt信号通路相关蛋白（Akt和PTEN）及凋亡促进基因（caspase-9）的表达情况。结果在10~100μmol/L范围内,Melittin可呈剂量和时间依赖的方式提高A549、SPC-A1细胞的增殖抑制率,差异均有统计学意义（P〈0.05）,但对16HBE无细胞毒性作用（P〉0.05）;与0μmol/L比较,除10μmol/L Melittin处理24 h后的晚期凋亡率和G2/M期细胞比例无统计学差异（P〉0.05）,10~100μmol/L的早、晚期凋亡率、G0/G1期细胞比例及PTEN和caspase-9蛋白水平均升高,S期、G2/M期细胞比例及Akt水平均降低,以上差异有统计学意义（P〈0.05）,且10~100μmol/L范围内各浓度间的差异均有统计学意义（P〈0.05）。结论 Melittin可对NSCLC细胞有毒性作用,但对正常肺上皮细胞无影响,且可诱导A549细胞凋亡及细胞周期阻滞并抑制PI3K/Akt信号通路的激活。

rhuPAa-melittin在卵巢癌体内外的作用机制

目的利用流式细胞术检测不同浓度的rhu PAa-melittin作用于卵巢癌细胞的活性,进而初步探讨rhu PAa-melittin对卵巢癌细胞的抑制作用及体内抗瘤作用。方法利用不同浓度的rhu PAa-melittin蛋白分别作用于人卵巢癌细胞48 h,利用流式细胞术检测细胞周期及凋亡。rhu PAa-melittin蛋白作用于卵巢癌皮下移植瘤模型,观察移植瘤的生长情况。结果分别以0、4、8、16μg/m L浓度作用人卵巢癌细胞48 h,凋亡率分别为1.16%、3.83%、6.51%、10.2%。不同浓度下G0/G1期细胞无明显作用,主要作用于S期,阻止由S期进入G2/M期,从而抑制肿瘤生长。结论 rhu PAa-melittin在卵巢癌的体内外实验中有效抑制了肿瘤的生长。

Melittin induces human gastric cancer cell apoptosis via activation of mitochondrial pathway

AIM: To investigate the apoptotic effects of melittin on SGC-7901 cells via activation of the mitochondrial signaling pathway in vitro.METHODS: SGC-7901 cells were stimulated by melittin, and its effect on proliferation and apoptosis of was investigated by methyl thiazolyl tetrazolium assay, morphologic structure with transmission electron microscopy, annexin-V/propidium iodide double-staining assay, measuring mitochondrial membrane potential(MMP) levels, and analyzing reactive oxygen species(ROS) concentrations were analyzed by flow cytometry. Cytochrome C(Cyt C), apoptosis-inducing factor(AIF), endonuclease G(Endo G), second mitochondria-derived activator of caspases(Smac)/direct IAP binding protein with low isoelectric point(Diablo), and FAS were analyzed by western blot. The expression of caspase-3 and caspase-8 was measured using activity assay kits.RESULTS: Melittin was incubated at 1.0, 2.0, 4.0, or 6.0 μg/m L for 1, 2, 4, 6, or 8 h and showed a timeand concentration-dependent inhibition of SGC-7901 cell growth. Melittin induced SGC-7901 cell apoptosis, which was confirmed by typical morphological changes. Treatment with 4 μg/m L melittin induced early apoptosis of SGC-7901 cells, and the early apoptosis rates were 39.97% ± 3.19%, 59.27% ± 3.94%, and 71.50% ± 2.87% vs 32.63% ± 2.75% for 1, 2, and 4 h vs 0 h(n = 3, P < 0.05); the ROS levels were 616.53% ± 79.78%, 974.81% ± 102.40%, and 1330.94% ± 93.09% vs 603.74% ± 71.99%(n = 3, P < 0.05); the MMP values were 2.07 ± 0.05, 1.78 ± 0.29, and 1.16 ± 0.25 vs 2.55 ± 0.42(n = 3, P < 0.05); caspase-3 activity was significantly higher compared to the control(5492.3 ± 321.1, 6562.0 ± 381.3, and 8695.7 ± 449.1 vs 2330.0 ± 121.9), but the caspase activity of the non-tumor cell line L-O2 was not different from that of the control. With the addition of the caspase-3 inhibitor(Ac-DEVD-CHO), caspase-3 activity was significantly decreased compared to the control group(1067.0 ± 132.5 U/g vs 8695.7 ± 449.1 U/g). The expression of the Cyt C, Endo G, and AIF proteins in SGC-7901 cells was significantly higher than those in the control(P < 0.05), while the expression of the Smac/Diablo protein was significantly lower than the control group after melittin exposure(P < 0.01). Ac-DEVD-CHO did not, however, have any effect on the expression of caspase-8 and FAS in the SGC-7901 cells.CONCLUSION: Melittin can induce apoptosis of human gastric cancer(GC) cells through the mitochondria pathways, and it may be a potent agent in the treatment of human GC.

蜂毒溶血肽(Melittin)cDNA的克隆及序列分析

为了克隆得到正确序列的蜂毒溶血肽 c DNA,对蜂毒中的蜂毒溶血肽进行了研究。实验从蜜蜂毒腺中提取总 RNA,通过 RT- PCR方法扩增得到了蜂毒溶血肽的 c DNA,扩增产物克隆到p T7Blu- T载体 ,转化大肠杆菌 JM1 0 9,DNA序列分析结果表明克隆得到的蜂毒溶血肽 c

3D-QSAR Study of Melittin and Amoebapore Analogues by CoMFA and CoMSIA Methods

Peptides are one of the indispensable substances in life. The use of computer aided drug design(CADD) methods to design peptides and peptiodmimetics can short the design cycle, save research funding, improve the level of whole research to a large extent and guide the discovery of new drugs. In this paper, Melittin and amoebapore three-dimensional quantitative structureactivity relationship(3D-QSAR) models were established by using comparative molecular field analysis(CoMFA) and comparative molecular similarity indices analysis(CoMSIA) method. The result shows that, the correlation coefficient(q^2) was 0.583 and non-cross-validation correlation coefficient(r^2) was 0.972 for the melittin CoMFA model. The q^2 and r^2 were 0.630 and 0.995 for the best CoMSIA model, 0.645 and 0.993 for the amoebapore CoMFA model, and 0.738 and 0.996 for the best CoMSIA model. The statistical parameters demonstrated that the CoMFA and CoMSIA models had both good predictive ability and high statistical stability, and can provide theoretical basis for designing new high activity polypeptide drugs.

rhuPAa-melittin的生物活性检测

目的利用高纯度的rhuPAa-melittin进行人成纤维细胞和卵巢癌细胞进行活性检测,探讨rhuPAa-melittin对卵巢癌治疗作用及对正常细胞毒性作用。方法利用高纯度的rhuPAa-melittin蛋白分别作用于人成纤维细胞和人卵巢癌细胞SKOV3 48 h,在490 nm波长处测定其吸光度,以反映活细胞的数量并间接反映rhuPAa-melittin对培养中细胞的生长抑制作用。结果人卵巢癌细胞SKOV3组,在4μg/mL时对细胞抑制率已经达到了66.59%,在16μg/mL时,细胞已经全部死亡,差异有统计学意义(P<0.05)。而人成纤维细胞组,在4μg/mL时其对细胞的抑制率仅为达到7.3%,8μg/mL时抑制率为12.3%,16μg/mL时抑制率为22.0%。结论 rhuPAa-melittin对人卵巢癌细胞SKOV3细胞有着明显的抑制杀伤作用,而对于人成纤维细胞的抑制作用不明显。

Expression and Purification of the Melittin Gene from Polistes hebraeus by the GST Gene Fusion System

A cDNA encoding melittin from a wasp (Polistes hebraeus) was amplified and cloned into the GST fusion expression vector pGEX-4T-2. The expressed protein appeared on the SDS-PAGE profiles with an about 29 kDa band. Western blotting proved that the recombinant protein is the fusion protein of GST-PhM. The expression conditions of GST-PhM fusion protein for E. coli BL21 transformant were optimized. Thin layer scanning on the SDS-PAGE profiles showed that the expressed target protein had accumulated up to about 10%～12% of total protein of bacterial cells under the optimized expression condition. Purified and recovered recombinant melittin of Polistes hebraeus showed bioactivity in activating rabbit platelets to aggregate.

牛乳铁蛋白活性肽（LactoferricinB）（3—17）与蜂毒肽（Melittins）（6—12）杂合多肽活性功能的测定

抗菌肽近年逐渐成为研究的热点。将牛乳铁蛋白活性多肽（LfcinB）与蜂毒肽（Melittins）核心功能区间，即LfcinB的3-17位的15个氨基酸与Melitins的6-12位的8个氨基酸，固相合成新的多肽分子Lfb15-Me8。经RT-HPLC和质谱检测合成多肽分子准确，纯度达到98％以上。经体外抑菌实验证明，所合成的新型抗菌肽分子对大肠杆菌ATCC25922、金黄色葡萄球菌ATCC25923、绿脓杆菌ATCC27853、酵母GS115、沙门氏菌ATCC12291的最小抑菌浓度（MIC）分别为16μg·mL^-1、32μg·mE-164μg·mL^-1、128μg·mL^-1、16μg·mL^-1，并在其抗菌浓度下并未表现溶血特性。获得了具有活性的新型多肽序列，为抗菌肽的研发奠定了新的研究内容。

Low molecular fucoidan and its macromolecular complex with bee venom melittin

Low molecular weight (LMW) fucoidan, obtained by free radical depolymerization of high molecular polysaccharide extract of brown algae Hizikia fusiformis, was complexed with HPLC purified bee venom melittin. Water soluble form of the LMW fucoidan – melittin complex shows increased anti-inflammatory activity, inhibiting the production of nitric oxide in murine macrophage cell line Raw 264.7. The LMW fucoidan:melitin complex obtained in this study showed good biological activities, resulting in 2-fold reduction of the melittin toxicity. The fucoidan: melittin macromolecular complex obtained should be useful in future therapeutic applications.

rhuPAa-melittin发酵条件及大规模纯化方法

目的 通过rhuPAa-melittin在80 L发酵罐中的发酵表达,优化发酵条件及改进适合大规模纯化的方法,获得纯化的目的蛋白.方法 采用实验制备的rhuPAa-melittin重组质粒,通过电转化法将rhuPAa-melittin-pPICZαC载体转入毕赤酵母（Pichia pastoris）中,用PCR法扩增和检测rhuPAa-melittin基因在毕赤酵母基因组中的整合.利用毕赤酵母进行小量发酵,摸索最佳表达时间、pH及最佳的纯化方法.通过rhuPAa-melittin在80 L发酵罐中的发酵表达,优化发酵条件及改进适合大规模纯化的方法.结果 rhuPAa-melittin发酵条件在温度为28℃～30℃,FM21培养基中添加0.5％的蛋白胨,甲醇最终的流加速度控制在9 mL/（h.L）,初始发酵液体积、DO值（溶解氧）在25％～ 30％,发酵时间为12h,pH在6.0～7.0时获得高产量、高纯度、高收率的蛋白.结论 获得产量约350 mg/L,纯度可达96％以上,收率可达65％左右的rhuPAa-melittin蛋白.

蜂毒肽Melittin对小鼠红细胞溶血效应及影响机制分析

蜂毒肽Melittin是一种具有高效抗细菌、真菌、肿瘤细胞等多种生物学活性的抗菌肽,然而其溶血活性限制了其发展为高效抗菌抗癌药物的应用.本文深入分析了Melittin对小鼠红细胞的作用机制,探讨影响Melittin溶血活性的细胞机制.结果表明,Melittin可以引起红细胞膜上形成空洞,抑制细胞内ATPase的活性.红细胞膜中糖蛋白或糖脂糖链中的唾液酸参与Melittin的相互作用,介导Melittin的溶血活性.质膜胆固醇也影响Melittin与红细胞膜相互作用,降低溶血活性.研究还发现,外源添加D-葡萄糖和D-蔗糖能显著抑制Melittin的溶血活性.研究结果为降低抗菌肽溶血性以及推动抗菌肽的药物应用提供了重要理论基础.

蜂毒肽突变体Melittin-K1抑制人肝癌细胞BEL-7402生长的作用

目的:为了提高蜂毒肽Melittin的抗肿瘤活性,我们设计出新型多肽并将其命名为Melittin-K1。并对多肽Melittin-K1抑制人肝癌细胞BEL-7402生长的作用进行研究。方法:采用CCK-8法检测细胞活力。扫描电镜拍照结合培养基上清中乳酸脱氢酶(lactic dehydrogenase,LDH)的表达水平来评价细胞膜的损伤水平。构建BEL-7402细胞裸鼠异种移植瘤模型来评价Melittin-K1、Melittin体内对肝癌生长的影响以及毒性。结果:与Melittin相比,Melittin-K1抑制BEL-7402细胞生长的作用更显著;与L02细胞相比,BEL-7402对Melittin-K1的杀伤作用更敏感;Melittin-K1体外抑制BEL-7402细胞生长呈时间、剂量依赖性。扫描电镜以及LDH检测结果均显示Melittin-K1处理组细胞膜的破损情况严重。BEL-7402细胞裸鼠皮下移植瘤模型结果表明,Melittin-K1体内显著抑制肿瘤生长,其抗肿瘤作用明显高于同剂量Melittin。Melittin-K1和Melittin体内毒性小,主要表现在,与模型组相比,体重曲线、肝功指标均未发生明显改变。结论:Melittin-K1能够明显抑制肝癌细胞BEL-7402的增殖,是一种极具潜力的抗肝癌药物。

Promoting effect of ethanol on dewetting transition in the confined region of melittin tetramer

To study the influence of ethanol molecules on the melittin tetramer folding,we investigated the dewetting transition of the melittin tetramer immersed in pure water and 8%aqueous ethanol solution(mass fraction) by the molecular dynamics simulations.We found that the marked dewetting transitions occurred inside a nanoscale channel of the melittin tetramer both in pure water and in aqueous ethanol solution.Also,ethanol molecules promoted this dewetting transition.We attributed this promoting effect to ethanol molecules which prefer to locate at the liquidvapor interface and decrease the liquid-vapor surface energy.The results provide insight into the effect of ethanol on the water dewetting phenomena.

Anti-angiogenesis effect of melittin on Mock/MHCC97-H cells by the regulation of cathepsin S in vivo

目的：通过体内实验探究蜂毒素通过调控组织蛋白酶S的表达抑制人肝癌Mock/MHCC97-H细胞肿瘤血管生成的作用.方法：分别建立人肝癌Mock/MHCC97-H细胞和沉默组织蛋白酶的shRNA-CatS/MHCC97-H细胞裸鼠原位移植瘤模型,模型小鼠随机分组共4组,Al组：小鼠接种shRNA-CatS/MHCC97-H细胞并给予蜂毒素治疗;A2组：小鼠接种shRNA-CatS/MHCC97-H细胞给予生理盐水;B1组：小鼠接种Mock/MHCC97-H细胞给予蜂毒素治疗;B2组：小鼠接种Mock/MHCC97-H细胞给予生理盐水.A1和B1治疗组为腹腔内注射蜂毒素80mg/kg·d;A2和B2组为腹腔内注射生理盐水0.2mL/d.给药25天,用药结束后隔天处死动物.观察并计录各组裸鼠肿瘤的大小和瘤重,并将取出的肝移植瘤组织进行CD34蛋白的免疫组化染色,用Westernblot检测蜂毒素对裸鼠肝移植瘤中组织蛋白酶S、VEGF-A、p-VEGFR2、Ras、Raf、p-Raf、MEK1、p-MEK1、ERK1/2和p-ERK1/2蛋白的作用.结果：与B2组相比,B1组的肿瘤体积明显缩小,重量明显减轻（均P〈0.001）;而A1组和A2组在肿瘤体积和重量相比,均无明显差异.B2组肿瘤的中CD34阳性微血管数目明显多于A2组（P〈0.001）,A1组肿瘤的中CD34阳性微血管数目明显多于B1组（P〈0.001）.蜂毒素能明显的抑制接种Mock/MHCC97-H细胞模型组组中CatS、VEGF-A、p-VEGFR2、Ras、Raf、p-Raf、MEK1、p-MEK1、ERK1/2和p-ERK1/2蛋白的表达,而对接种shRNA-CatS/MHCC97-H细胞模型组的上述蛋白的表达无明显作用.结论：蜂毒素可以通过抑制组织蛋白酶S引导的VEGF-A信号通路,进而抑制组织蛋白酶S诱发的肿瘤生长和新生血管生成.

Melittin enhanced intracellular trehalose with synergistic membrane stabilization of macromolecular protectants for cell cryopreservation

Cryopreservation plays an essential role in biobanking and cell therapy,but the physiological toxicity of traditional cryoprotectants such as glycerol and dimethyl sulfoxide(DMSO)has raised safety issues for biomedical applications.Trehalose,a nonreducing disaccharide that accumulates in desiccation-or cold-tolerant organisms,has been considered as a biocompatible cryoprotectant.Herein,a naturally occurring membrane-active cationic peptide,melittin,was utilized to facilitate membraneimpermeable trehalose entry into cells for effective cell cryopreservation.Poly(ethylene glycol)and poly(vinyl pyrrolidone)were applied as macromolecular protectants to improve the stabilization of cell membranes.Upon the optimal protocol,the postthaw recovery of human red blood cells in freezing bags at a hematocrit of~50%could achieve 82.9%with favorable cell morphologies and physiological functions.Furthermore,the cryosurvival of L929 fibroblasts reached 84.3%compared to the conventional method using 10%(v/v)DMSO.In short,this work by using trehalose and melittin provides a biocompatible solvent-free approach for long-term cryostorage of cells.

Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency

Objective Melittin and its derivatives have been characterized to establish effective gene delivery systems.Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery.Nevertheless,little investigation has been conducted to explore its potential application in the context of viral vectors.Methods Various melittin-derived peptides were inserted into the loop VIII of the capsid proteins of recombinant adeno-associated virus vectors.These vectors carrying either gfp or fluc genes were subjected to qPCR assays and transduction assays of HEK293T cells to investigate the efficiency of vector production and gene delivery.In addition,the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice.Finally,the intricate details of the vector-mediated transduction mechanisms were revealed by specific pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle.Results A total of 76 melittin-related peptides were compiled from existing literature.Among them,cMA2,Melt13,p5RHH and aAR3 were found to significantly enhance the gene delivery efficiency of rAAV2 vectors.The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV.Mechanistically,bafilomycin A1,a vacuolar endosome acidification inhibitor,completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors.Most importantly,p5RHH-rAAV8 vectors also demonstrated increased hepatic transduction in vivo in C57BL/6 mice.Conclusion The incorporation of melittin analogues into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression.While further modifications remain an area of interest,our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery.

The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo

Objective: Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus(rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based r AAV2 vector delivery strategy.Methods: The melittin peptide was inserted into the rAAV2 capsid either in the loop Ⅷ of all viral proteins(VPs) or at the N terminus of VP2. Various r AAV2-gfp or-fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression.Results: rAAV2 vectors with melittin peptide inserted in the loop Ⅷ of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of r AAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity.Conclusion: Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

Melittin-IL-2融合蛋白的抗肿瘤活性

蜂毒素（Melittin）是欧洲蜜蜂（Apis mellifera）蜂毒中的主要成分，为26个氨基酸组成的碱性多肽。蜂毒疗法是我国利用传统中医中药治疗肿瘤的独特方法。Melittin具有抗菌、抗肿瘤、抗关节炎、抗艾滋病病毒等诸多生物学活性，还具有免疫调节功能，并可对多种肿瘤细胞有直接杀伤作用。白细胞介素-2（interleukin-2，IL-2）具有多种与免疫功能强化有关的生物学活性。其制剂及融合蛋白已有研究报道并取得了一定的效果。

意大利蜜蜂毒Melittin组分的研究（Ⅰ）──分离纯化及理化性质研究

用ＳｅｐｈａｄｅｘＧ─２５、ＳｅｐｈａｄｅｘＧ─５０、ＣｅｌｌｕｏｓｅＣＭ３２三步层析从意大利蜜蜂毒中分离纯化得到对家兔血小板具有强诱导聚集作用的Ｍｅｌｉｔｔｉｎ活性组分，它在ＳＤＳ─聚丙烯酰胺凝胶电泳图谱上呈一均匀蛋白带，分子量为３０００，由２６个氨基酸残基组成。Ｍｅｌｉｔｔｉｎ诱导家兔血小板聚集的最适剂量为６０～８０μｇ／ｍｌ；对热较稳定；在酸性环境中活性被抑制，在碱性环境中活性较高；Ｃｕ￣（２＋）对其活性具有明显抑制作用。